Isolation of differentially expressed genes upon immunoglobulin class switching by a subtractive hybridization method using uracil DNA glycosylase.
Immunoglobulin class switch recombination enables B lymphocytes to sequentially express antibodies that have identical specificities but that differ in class and effector function. Although several cis elements required for class switch recombination have been identified, few trans -acting factors which are directly related to class switching have been found. Previously we have developed an efficient in vitro class switching system using a cell line, CH12F3-2. To clarify the molecular mechanism of class switching, we intended to isolate genes induced with interleukin (IL)-4, transforming growth factor (TGF)-beta and CD40L using the in vitro class switching system. For that purpose, an improved method for making subtracted cDNA libraries, using uracil DNA glycosylase, has been developed. This method can overcome a general problem of subtraction, that rare cDNAs are easily lost. This new subtraction method was applied to the CH12F3-2 switching system to isolate genes induced by stimulations with IL-4, TGF-beta and CD40L, and cDNAs encoding deiodinase 1 and SS32, an alternatively spliced form of the muscle LIM protein, were obtained. Their expression patterns in response to various combinations of stimuli and the time courses of the induction supported the usefulness of this method.
Sugai
M
M
Department of Medical Chemistry, Faculty of Medicine and Center for Molecular Biology and Genetics, Kyoto University Sakyo-ku, Kyoto 606, Japan.
Kondo
S
S
Shimizu
A
A
Honjo
T
T
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D045647:DNA Glycosylases; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D007134:Immunoglobulin Switch Region; D066298:In Vitro Techniques; D060588:LIM Domain Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics; D009699:N-Glycosyl Hydrolases; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics; D051981:Uracil-DNA Glycosidase
D017931:DNA Primers; D018076:DNA, Complementary; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; C090114:cysteine and glycine-rich protein 3; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase